System and method for retrieving and analyzing particles

ABSTRACT

A system and method for isolating and analyzing single cells, including: a substrate having a broad surface; a set of wells defined at the broad surface of the substrate, and a set of channels, defined by the wall, that fluidly couple each well to at least one adjacent well in the set of wells; and fluid delivery module defining an inlet and comprising a plate, removably coupled to the substrate, the plate defining a recessed region fluidly connected to the inlet and facing the broad surface of the substrate, the fluid delivery module comprising a cell capture mode.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/815,532, filed 16 Nov. 2017, which is a continuation-in-part of U.S.application Ser. No. 15/657,553, filed 24 Jul. 2017, which is acontinuation of U.S. patent application Ser. No. 15/333,420, filed 25Oct. 2016, which is a is a continuation of U.S. patent application Ser.No. 14/607,918, filed 28 Jan. 2015, which is a continuation of U.S.patent application Ser. No. 13/557,510, filed 25 Jul. 2012, and claimsthe benefit of U.S. Provisional Application Ser. No. 61/513,785 filed on1 Aug. 2011, which are all incorporated in their entirety by thisreference.

This application is also a continuation-in-part of U.S. application Ser.No. 15/720,194, filed 29 Sep. 2017, which is a continuation ofco-pending U.S. application Ser. No. 15/431,977, filed 14 Feb. 2017,which is a continuation of U.S. application Ser. No. 14/863,191 (nowU.S. Pat. No. 9,610,581), filed 23 Sep. 2015, which is a continuation ofU.S. application Ser. No. 14/208,298 (now U.S. Pat. No. 9,174,216),filed 13 Mar. 2014, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/894,150, filed on 22 Oct. 2013, U.S. ProvisionalApplication Ser. No. 61/829,528, filed on 31 May 2013, and U.S.Provisional Application Ser. No. 61/779,049, filed on 13 Mar. 2013,which are all incorporated herein in their entirety by this reference.

This application is also a continuation-in-part of U.S. application Ser.No. 15/430,833, filed 13 Feb. 2017, which is a continuation of U.S.patent application Ser. No. 15/199,245, filed 30 Jun. 2016, which is acontinuation of U.S. patent application Ser. No. 14/208,458, filed 13Mar. 2014, which claims the benefit of U.S. Provisional Application Ser.No. 61/902,431, filed on 11 Nov. 2013, and U.S. Provisional ApplicationSer. No. 61/779,090, filed on 13 Mar. 2013, all of which areincorporated herein in their entirety by this reference.

This application claims the benefit of U.S. Provisional Application Ser.No. 62/423,322, filed 17 Nov. 2016, and U.S. Provisional ApplicationSer. No. 62/545,251, filed 14 Aug. 2017, each of which is incorporatedherein in its entirety by this reference.

This application is also related to U.S. application Ser. No.15/442,222, filed 24 Feb. 2017, which is incorporated herein in itsentirety by this reference.

TECHNICAL FIELD

This invention relates generally to the particle analysis field, andmore specifically to a new and useful system and method for retrievingand analyzing particles within the particle analysis field.

BACKGROUND

With an increased interest in cell-specific drug testing, diagnosis, andother assays, systems that allow for individual cell isolation,identification, and retrieval are becoming more desirable within thefield of cellular analysis. Furthermore, with the onset of personalizedmedicine, low-cost, high fidelity cellular analysis systems are becominghighly desirable. However, preexisting cell and other particle capturesystems suffer from various shortcomings that prevent widespreadadoption for cell-specific testing. For example, flow cytometry requiresthat the cell be simultaneously identified and sorted, and limits cellobservation to the point at which the cell is sorted. Flow cytometryfails to allow for multiple analyses of the same cell within a singleflow cytometry workflow, and does not permit arbitrary cellsubpopulation sorting. Conventional microfluidic devices typically failto allow for subsequent cell removal without cell damage, and onlycapture the cells expressing the specific antigen; non-expressing cells,which could also be desired, are not captured by these systems. Suchloss of cell viability can preclude live-cell assays from beingperformed on sorted or isolated cells. Cellular filters can separatesample components based on size without significant cell damage, butsuffer from clogging and do not allow for specific cell identification,isolation of individual cells, and retrieval of identified individualcells. Other technologies in this field are further limited in theirability to allow multiplex assays to be performed on individual cells,while minimizing sample preparation steps and overly expensiveinstrumentation.

Thus, there is a need in the particle sorting field to create new anduseful systems and methods for retrieving and analyzing cells, and theinventions disclosed herein provide such useful systems and methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representation of an embodiment of the system.

FIB. 1B is a schematic representation of an example of the system.

FIG. 2 depicts a first and second micrograph of cells extracted usingthe system.

FIG. 3A depicts specific examples of various aspects of the system.

FIG. 3B is a schematic representation of a variation of a methodperformed using the system.

FIGS. 4A-4B are perspective views of a first specific example of thesystem, with and without a particle receptacle, respectively.

FIG. 5A is a first detail view of a region of FIG. 1B.

FIG. 5B is a plan view of a specific example of particle capturesubstrates and a particle receptacle arranged in the system.

FIGS. 6A-6B are a second and third detail view, respectively, of FIG.1B.

FIG. 7 is a perspective view of a second specific example of the system.

FIG. 8 is a flow chart representation of an embodiment of the method.

FIG. 9 is a schematic representation of a first example of the method.

FIG. 10 is a flow chart representation of a second example of themethod.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiments of the inventionis not intended to limit the invention to these preferred embodiments,but rather to enable any person skilled in the art to make and use thisinvention.

1. System.

As shown in FIG. 1A, a system 100 for retrieving and analyzing aparticle of a set of particles preferably includes: a structural frame10 supporting: a capture stage 110 that positions a particle capturesubstrate 200 in a capture mode of the system 100; an imaging subsystem120 operable to image the particle capture substrate 200 (e.g., whereinthe imaging subsystem 120 includes an illumination subsystem 122operable to transmit light toward the capture stage 110 and cooperatingwith an optical sensor 126 operable to generate an image dataset ofcontents of the particle capture substrate 200 in the capture mode); aparticle retriever subsystem 130 including a pump 132 fluidly coupled toa particle extractor 134; an actuation subsystem 140 including a firstunit 141 coupled the capture stage, a second unit 142 coupled to theimaging subsystem, and a third unit 143 coupled to the particleretriever subsystem; a particle receptacle station 150 hosting aparticle receptacle 152; and a control subsystem 160 that, based on aposition of the particle at the capture substrate identified from theimage dataset, generates commands to retrieve the particle and transmitthe particle to the particle receptacle 152.

In more detail (e.g., as shown in FIGS. 1B and/or 7), an embodiment ofthe system 100 can include: a structural frame 10 supporting: a capturestage 110 defining a broad face 112 that positions a particle capturesubstrate 200 having a set of particle capture chambers 210 orientedperpendicular to the broad surface 112 in a capture mode of the system100, the broad surface 112 including an opening 114 toward a closedsurface 220 of the capture substrate 200 in the capture mode; an imagingsubsystem 120 including: an illumination subsystem 122 operable totransmit light toward the opening 114, a filter subsystem 124 operableto filter light transmitted between the illumination subsystem 122 andthe capture substrate 200 in the capture mode, and an optical sensor 126cooperating with a focusing and optics subsystem 128 that manipulateslight transmitted to the optical sensor 126, the optical sensor 126operable to generate an image dataset of contents of the set of particlecapture chambers 210 in the capture mode; a particle retriever subsystem130 including a pump 132 fluidly coupled to a particle extractor 134(e.g., capillary tube) having a capture end 135 facing an open surface230 of the capture substrate in the capture mode; an actuation subsystem140 including a first unit 141 coupled to the capture stage, a secondunit 142 coupled to the imaging subsystem, and a third unit 143 coupledto the particle retriever subsystem; a particle receptacle station 150hosting a particle receptacle 152; and a control subsystem 160 that,based on a position of the particle at the capture substrate identifiedfrom the image dataset, generates commands for aligning the particleextractor 134 with the position of the particle by the actuationsubsystem, controlling pressure provided by the pump of the particleretriever subsystem, and transmitting the particle to the particlereceptable, thereby retrieving the particle in single-particle format inthe capture mode.

In some variations, the system 100 can include: a display 170 incommunication with the control subsystem 160, the display operable torender at least one of: control parameters of the system associated withthe control subsystem 160 and images derived from the image dataset; anda containment subsystem 180 (e.g., sterile hood) operable to create asterile environment for sample handling, the containment subsystem 180configured about the structural frame 10.

The system preferably functions to provide a portable, sterileenvironment for retrieval of individual cells (e.g., captured insingle-cell format) and/or cell clusters. The system preferably enablesautomated cell localization and identification (e.g., based on imagedata, such as fluorescence microscopy data), cell extractor 134 (e.g.,capillary tube) alignment and insertion, cell extraction (e.g., byaspiration), and/or extracted cell delivery (e.g., to a specifiedlocation of a cell receptacle such as a multi-well plate). However, thesystem can additionally or alternatively perform any other suitablefunctions.

In a specific example, with a capillary having a 30 μm inner diameter(or other suitable diameter), the system can achieve a throughput ofover 90 particles retrieved in 90 minutes, transferring retrieved singlecells in a viable state to downstream containers such as well plates(e.g., 96-well plates, well plates of any other suitable format), tubes(e.g., PCR tubes, conicals, etc.), dishes (e.g., Petri dishes), or anyother suitable downstream container. As shown in FIG. 2, cells (e.g.,MCF7 cells) retrieved in single-cell format from the system can beretrieved in a viable state and grown in culture for further analysis.In the specific example, 5-color fluorescence and brightfield imagingsubsystems of the system facilitate positioning of the capillaryrelative to a target particle/cell of interest for retrieval, incoordination with a computing system for image acquisition, control ofillumination, and imaging focus. In the specific example, the system canbe placed in a sterile hood for sterile operation; however, variationsof the system can alternatively have any other suitable dimension(s) inrelation to sterile sample processing.

The structural frame 10 preferably functions to support the otherelements of the system 100 (e.g., mechanically coupled to the otherelements, such as statically coupled and/or coupled via one or moreactuator and/or hinged). For example (e.g., as shown in FIG. 1B), thestructural frame 10 can include a member supporting some or all of thefocusing and optics subsystem 128 (e.g., by way of a second unit 142 ofthe actuation subsystem 140), a member supporting the remainder of theimaging subsystem 120, a member supporting the capture stage 110 (e.g.,by way of a first unit 141 of the actuation subsystem 140) andoptionally the particle receptacle station 150, and a member supportingthe particle retriever subsystem 130 (e.g., by way of a third unit 143of the actuation subsystem 140).

In some embodiments, the structural frame member supporting the particleretriever subsystem 130 includes an arm extending between the actuationsubsystem third unit 143 and the particle retriever subsystem 130 (e.g.,cantilevered from the third unit 143), which preferably retains theparticle extractor substantially in alignment with the optical axis. Theare is preferably configured to minimize undesired particle extractormotion (e.g., due to vibration), such as limiting such motion to lessthan a threshold deviation from the desired position (e.g., less than10, 5, 2, 1, 0.5, 0.25, or 0.1 microns). For example, the arm can have anatural vibrational mode resulting in a vibrational amplitude of lessthan the threshold deviation, and/or the system can include one or morevibration dampers between the arm and the third unit 143 (e.g., therebyreducing particle extractor vibrational motion). However, undesiredparticle extractor motion can additionally or alternatively be minimizedin any other suitable manner.

In some embodiments, the structural frame 10 includes multipleindependent frame modules (e.g., each supporting elements of distinctsubsystems) configured to be attached (e.g., reversibly and/orrepeatably attached) to each other, such as by mechanical fasteners(e.g., bolts, clips, clamps, etc.). For example (e.g., as shown in FIGS.4A-4B), the structural frame 10 can include a first module that supports(e.g., houses, encloses, etc.) the imaging subsystem 120 (and secondunit 142 of the actuation subsystem 140) and a second module thatsupports the capture stage, particle retriever subsystem 130, andremaining elements of the actuation subsystem 140 (e.g., wherein thesecond module has dimensions between 10 and 50 cm on each side, such as33×41×25 cm).

The structural frame 10 preferably includes (e.g., is made of) one ormore rigid materials, such as metal and/or a rigid polymer. Thestructural frame 10 can optionally enclose (or substantially enclose)all of some of the other elements of the system (e.g., thereby providingmechanical protection for the elements and/or otherwise isolating theelements from their surroundings). For example, the structural frame 10can form an optical enclosure (e.g., opaque enclosure, such as a full orpartial light-tight enclosure) around some or all of the imagingsubsystem 120 (e.g., reducing background readings from ambient light).However, the structural frame 10 can additionally or alternativelyinclude any other suitable elements in any other suitable configuration.

1.1 Capture Stage.

The capture stage 110 preferably functions to receive and align one ormore particle capture substrates 200 (e.g., cell capture devices)relative to the imaging subsystem 120 (e.g., the illumination module110, focusing and optics subsystem 128, optical sensor 126, etc.), theparticle retriever subsystem 130 (e.g., the cell extractor 134), and/orany other suitable elements of the system 100 (e.g., as shown in FIGS.5A, 5B, and 6A). Such alignment can enable light-based analyses and/oroptically-guided retrieval of captured cells (and/or other particles) ofinterest within the particle capture substrate 200.

The capture stage 110 preferably defines a broad face 112 coupled to(e.g., retaining, supporting, etc.) one or more particle capturesubstrates 200. For example, the capture stage 110 can support aplurality of particle capture substrates 200 (e.g., a closed surface 220of each substrate 200 retained against the broad face 112 by gravity, byone or more fasteners such as spring clips and/or screws pressing uponan open surface 230 of each substrate 200, etc.). The capture stage 110preferably positions the particle capture substrate 200 such that abroad face of the substrate (e.g., the closed surface 220) is against(e.g., substantially coplanar with) the broad face 112 (e.g., in acapture mode). For example, the substrate 200 can be positioned suchthat a set of particle capture chambers 210 (e.g., defined in the opensurface 230, such as normal the open surface 230 and/or closed surface220) are oriented perpendicular to the broad surface 112. The capturestage 110 preferably does not impede access to the open surface 230(e.g., to the chambers 210), but can additionally or alternativelyinclude any suitable elements arranged on and/or near the open surface230.

The broad face 112 preferably includes one or more openings 114. Eachopening 114 can provide optical access (e.g., allow light transmission,enable close proximity of an objective lens, etc.) to the closed surface220 of a substrate 200. Each opening 114 can be a void defined in thebroad face 112, a window of transparent material, and/or can be anyother suitable opening 114.

The capture stage 110 can additionally or alternatively support theparticle receptacle station 150 (e.g., adjacent the particle capturesubstrates 200). In one example, the capture stage 110 rigidly couplesthe particle capture substrates 200 and the particle receptacle station150, enabling coordinated movement of the capture stage 110 and all therigidly coupled elements (e.g., by the first unit 141 of the actuationsubsystem 140). However, the capture stage 110 can additionally oralternatively support any other suitable elements of the system in anyother suitable manner.

The capture stage 110 can optionally include elements as described inU.S. application Ser. No. 15/430,833, filed 13 Feb. 2017 and titled“System for Imaging Captured Cells”, which is herein incorporated in itsentirety by this reference (e.g., as described regarding the platform).However, the capture stage 110 can additionally or alternatively includeany other suitable elements in any suitable arrangement.

1.2 Imaging Subsystem.

The imaging subsystem 120 preferably includes: an illumination subsystem122 (e.g., operable to transmit light toward the opening 114), a filtersubsystem 124 (e.g., operable to filter light transmitted between theillumination subsystem 122 and the capture substrate 200 in the capturemode), and an optical sensor 126 cooperating with a focusing and opticssubsystem 128 that manipulates light transmitted to the optical sensor126 (e.g., the optical sensor 126 operable to generate an image datasetof contents of the set of particle capture chambers 210 in the capturemode), such as shown in FIG. 6B. The imaging subsystem 120 can includeelements such as those described in U.S. application Ser. No.15/430,833, filed 13 Feb. 2017 and titled “System for Imaging CapturedCells”, which is herein incorporated in its entirety by this reference.

The imaging subsystem 120 preferably includes a microscope (e.g.,inverted microscope) such as a fluorescence microscope. In one example,the illumination subsystem 122 includes a bright-field illuminationsource (e.g., white light source such as one or more white LEDs,narrow-spectrum and/or single wavelength light source, etc.) and/or afluorescence light source (e.g., wide-spectrum light source, preferablyincluding ultraviolet and/or infrared wavelengths of light), preferablywith adjustable intensity; the filter subsystem 124 includes one or moreexcitation filters, emission filters, and/or dichroic mirrors (e.g.,grouped into one or more filter modules, such as aligned groupsincluding a single excitation filter, dichroic mirror, and emissionfilter); the optical sensor 126 includes a photodiode comprising aphotoelectric material configured to convert electromagnetic energy intoelectrical signals; and the focusing and optics subsystem 128 includes alens (e.g., objective lens) configured to focus light from theillumination module onto a target object (e.g., particle capturesubstrate 200, captured cell, capture end 135 of the cell extractor 134,etc.) at the capture stage 110 (and/or a lens configured to focus lightfrom the target object onto the optical sensor). The lens is preferablyoriented substantially normal the broad face 112 (e.g., defines anoptical axis substantially normal the broad face 112). The lens (and/orother elements of the focusing and optics subsystem 128) is preferablyconfigured to be moved (e.g., translated substantially along the opticalaxis) by the second unit 142 of the actuation subsystem 140. However,the imaging subsystem 120 can additionally or alternatively include anyother suitable elements in any other suitable arrangement.

1.3 Particle Retriever Subsystem.

The particle retriever subsystem 130 preferably functions to extract atleast one of a single cell and a cell cluster (and/or any other suitableparticles) from a well of the array. While an individual cell from asingle well is preferably selectively removed, the particle retrieversubsystem 130 can facilitate simultaneous multiple cell/cell clusterremoval from the set of wells. The cell/cell cluster is preferablyremoved by applying a removal force to the cell. The removal force canbe applied by capillary force, but can additionally or alternatively beapplied by aspirating the contents out of a well (i.e., using a negativepressure). The removal force can additionally or alternatively beapplied by pumping fluid through the set of wells (e.g., by way of aperimeter channel) to provide a positive pressure that drives thecell/cell cluster from the well. In one variation, the pump pressureprovided by a pump mechanism at the particle retriever subsystem 130 isless than 10,000 Pa, and in a specific variation, the provided pumppressure is 6,000 Pa. However, any other suitable pump or aspirationpressure can be used.

In some variations, the particle retriever subsystem 130 can comprise acell extractor 134. The cell extractor 134 functions to selectivelyremove one or more isolated cells from an addressable location withinthe system 100. The cell extractor 134 is preferably configured toremove a cell/cell cluster from a single well, but can alternatively beconfigured to simultaneously remove multiple cells/cell clusters frommultiple wells. The particle retriever subsystem 130 is preferablyoperable in an extraction mode, wherein in the extraction mode theparticle retriever subsystem 130 extracts at least one of a set ofsingle cells from a well of the set of wells, along a direction normalto the base surface of the well. In the extraction mode, the fluiddelivery module is preferably removed from the substrate; however, thefluid delivery module can alternatively remain coupled to the substratewhen the cell removal module is operated in the extraction mode.

In a first variation of the cell extractor 134, the cell extractor 134is configured to access the set of wells from a direction normal to theopen surface 220 (e.g., broad surface) of the substrate 200. The cellextractor 134 preferably removes the cell/cell cluster in asubstantially normal direction from the open surface 220 of thesubstrate 200, but can alternatively remove the cell/cell cluster in anangled direction relative to the open surface 220. The cell extractor134 preferably defines an interior void, such as a hollow channel (e.g.,of a micropipette, capillary tube such as a glass capillary tube, etc.),between a capture end 135 and an outlet (e.g., opposing the capture end135 across the length of the cell extractor 134) that accesses the setof wells and defines a substantially fluidly isolated volume in fluidcommunication with one or more wells. The void can include one or moresealing elements at the capture end 135 (e.g., a polymeric coating oradequate geometry) that facilitate fluid seal formation with the well(s)113. The particle retriever subsystem 130 can optionally include aprotective member (e.g., polymer sheath) surrounding a portion of thecell extractor 134 (e.g., surrounding most of an exposed length of theextractor 134, wherein the extractor tip emerges from the sheath toavoid sheath interference with the substrate 200 during tip insertion).The cell extractor 134 preferably tapers from a proximal end to thecapture end 135 (e.g., tip), in order to provide an adequate geometry toreceive contents of a well into the cell extractor 134; however, thecell extractor 134 can alternatively have any other suitable form. Assuch, the hollow needle is preferably configured to form a substantiallyfluidly isolated volume within a well of interest, and a low-pressuregenerator (e.g., a pump) is then used to aspirate the retained cell/cellcluster out of the well, through the hollow channel, and into a cellcollection volume of the cell extractor 134. The void preferably definesa micron-scale aperture at the capture end 135, such as an aperturehaving a characteristic dimension (e.g., diameter, width, inscribedand/or circumscribed circle diameter, etc.) between 1 micron and 500microns (e.g., between 10 and 100 microns, between 20 and 50 microns, 30microns, 40 microns, etc.). In one variation, the cell extractor 134 isa micropipette having a height of 200 micrometers and a hollow channeldiameter of 25 micrometers; in another variation, the cell extractor 134is a capillary tube having a channel diameter of 30 micrometers; in athird variation, the cell extractor 134 is a capillary tube having achannel diameter of 150 micrometers. In another variation, the wells ofthe set of wells are grouped such that each group may be circumscribedby a closed curve in the plane parallel to the broad surface of thesubstrate, and the cell extractor 134 has an inner diameter that issmaller than the largest chord of the closed curve. In anothervariation, the inner diameter is smaller than a characteristic dimension(e.g., width, diameter, etc.) of a single well. However, othervariations of these specific examples can have any other suitabledefining dimensions.

The cell extractor 134 can enable aspiration and/or dispensal of asamples (e.g., particles such as cells, surrounding liquid, etc.) up toa maximum volume (e.g., equal to or less than the volume of the void ora Tillable portion thereof). The maximum volume can be a volume between0.1 and 500 microliters (e.g., between 1 and 50 microliters, such as 5,10, or 25 microliters). However, the cell extractor 134 can additionallyor alternatively accommodate any other suitable sample volume.

The cell extractor 134 can be manufactured using microfabricationtechniques, or can additionally or alternatively be injection molded,laser cut, stamped, or manufactured using any other suitablemanufacturing technique. In one variation of hollow needle manufacture,a lumen is preferably etched into a substrate, such as silicon, usingetching techniques such as deep reactive ion etching (DRIE), plasmaetching, or any other suitable etching method. This step is preferablyutilized with a mask that covers the portions of the substrate 105 to beprotected. The walls and associated profiles are then preferablymanufactured through isotropic etching of the substrate 105 utilizing acorrosive liquid or plasma, but any other suitable isotropic materialremoval method can be used. A mask is preferably used to protect thepuncture end. In a second variation, tubes (e.g., glass tubes, plastictubes, etc.) can be pulled (e.g., by applying controlled heating to thetube end and pulling the tube under controlled tension) to narrow thetube opening to the desired diameter. Multiple hollow needles arepreferably simultaneously manufactured as an array 200, but canalternatively be individually manufactured.

The particle retriever subsystem 130 preferably includes a pump 132configured to alter pressure within the cell extractor void (e.g.,within the hollow channel of the capillary tube). The pump 132 ispreferably a positive displacement pump, more preferably a syringe pump,but can additionally or alternatively include any other suitablepump(s). For example, the pump 132 can include a piezoelectric actuator,a diaphragm pump, and/or any other suitable pumping mechanisms.

The pump 132 is preferably controlled by a pump actuator 144, morepreferably a motorized actuator (e.g., configured to be controlled bythey control subsystem 160), such as described below regarding theactuation subsystem 140. However, the pump 132 can additionally oralternatively be controlled directly (e.g., by manual translation of thesyringe pump plunger within the syringe pump barrel, such as by pushingor pulling directly on the plunger by hand).

The pump 132 (e.g., a fluid port of the pump, such as an inlet oroutlet) is preferably fluidly coupled to the cell extractor 134 (e.g.,to the void) by a tube 136, more preferably a flexible tube. A flexibletube can enable independent movement of the cell extractor 134 withrespect to the pump 132 (e.g., during actuation of the actuationsubsystem third unit 143; such as during alignment, insertion, and/orremoval of the capture end 135). The tube 136 preferably includes (e.g.,is made of) a polymeric material (e.g., Teflon, Tygon, polyethylene,etc.), but can additionally or alternatively include metal (e.g., steel,copper, etc.) and/or any other suitable materials. To create effectivepumping pressure (e.g., for cell extraction), the dead-volume of thetube 136 is preferably minimized, such as a dead-volume less than athreshold maximum volume (e.g., less than 25, 15, 10, 5, 2, 1, or 0.5microliters). In one example, the dead-volume is reduced by placing a afiller element, such as a wire, inside the tube (e.g., 400 microndiameter wire placed within a tube with a 500 micron inner diameter),thereby occupying a portion of the tubing volume. The tube 136 ispreferably a single tube running between the pump 132 and cell extractor134, but can additionally or alternatively include any suitable fluidmanifold and/or other fluidic coupling.

The particle retriever subsystem 130 preferably enables easy removaland/or attachment (e.g., reattachment) of the cell extractor 134 (e.g.,capillary tube). This can enable cell extractor cleaning and/orreplacement (e.g., of contaminated and/or damaged cell extractors). Forexample, the cell extractor 134 can be coupled to the tube 136 by afriction fitting (e.g., optionally including hose barbs defined on thecell extractor 134 and/or hose clamps retaining the tube 136 in place onthe cell extractor 134). The particle retriever subsystem 130 caninclude a number of disposable (e.g., one-time use) cell extractors 134,and/or can include one or more cell extractors 134 configured for reuse.However, the particle retriever subsystem 130 can include any othersuitable set of cell extractors 134 of any suitable type(s), and/or caninclude only a single cell extractor 134 (e.g., non-removeable cellextractor).

The particle retriever subsystem 130 can, however, include any othersuitable cell removal tool, such as that described in U.S. applicationSer. No. 13/557,510, entitled “Cell Capture System and Method of Use”and filed on 25 Jul. 2012, which is herein incorporated in its entiretyby this reference.

Cell removal from the system 100 is preferably automated, but canadditionally or alternatively be semi-automated or manual. Furthermore,cell removal can be performed along with cell identification, comprisingautomatic fixing, permeabilization, staining, imaging, andidentification of the cells removed from the set of wells through imageanalysis (e.g., through visual processing with a processor, by using alight detector, etc.) or in any other suitable manner. The particleretriever subsystem 130 can be configured to facilitate advancement of acell extractor 134 to a well containing a cell/cell cluster of interest,for instance, with an actuation subsystem. The particle retrieversubsystem 130 can additionally or alternatively be configured tofacilitate cell removal method selection and/or cell removal toolselection. In another variation, cell identification at the particleretriever subsystem 130 can be semi-automated, and cell retrieval can beautomated. For example, cell staining and imaging can be doneautomatically, wherein identification and selection of the cells ofinterest can be done manually. In another variation, all steps can beperformed manually. However, any combination of automated or manualsteps can be used.

1.4 Actuation Subsystem.

The actuation subsystem 140 preferably includes a first unit 141 coupledto (e.g., controlling motion of) the capture stage 110, a second unit142 coupled to (e.g., controlling motion of) the imaging subsystem 120,a third unit 143 coupled to (e.g., controlling motion of) the particleretriever subsystem 130, and a pump actuator 143 coupled to (e.g.,controlling pumping action of) the pump 132.

The first unit 141 preferably enables and/or controls lateral motion(e.g., translation along one or more axes substantially parallel thebroad face 112) of the capture stage 110 and/or particle receptaclestation 150. For example, the first unit 141 can include an X-axistranslator (e.g., controlling lateral translation along a long edge ofthe capture stage 110) and a Y-axis translator (e.g., controllinglateral translation along an axis perpendicular to the X-axis). Thefirst unit 141 can additionally or alternatively enable and/or controltranslation along an out-of-plane axis (e.g., Z-axis substantiallyperpendicular the X- and Y-axes, axis substantially normal the broadface 112, axis substantially parallel the optical axis, vertical axis,etc.), lateral rotation (e.g., about the out-of-plane axis), tilt (e.g.,rotation about one or more axes substantially parallel the broad face112, such as the X- and/or Y-axis), and/or any other suitable motion.

The first unit 141 can optionally include an actuator for moving thecapture stage 110 and/or particle receptacle station 150 between aparticle extraction configuration (e.g., in which the particle capturesubstrate 200 is aligned with the particle extractor 134 and/or opticalaxis) and a particle delivery configuration (e.g., in which the particlereceptacle station 150 is aligned with the particle extractor 134 and/oroptical axis). For example, the capture stage 110 and particlereceptacle station 150 (and optionally, other actuators of the firstunit 141) can translate along a track and/or rotate about a joint axis(e.g., vertical axis, horizontal axis, etc.) of a cantilever arm toswitch between the particle extraction and particle deliveryconfigurations. However, the first unit 141 can additionally oralternatively include any other suitable elements in any other suitableconfiguration.

The second unit 142 preferably includes a focus actuator enabling and/orcontrolling imaging subsystem 120 focusing (e.g., by moving theobjective lens closer to and/or farther from the imaging target, such asthe particle capture substrate 200, its contents, and/or the capture end135 of the particle extractor). For example, the focus actuator canenable and/or control translation of the objective lens (and/or otheroptical elements) along the optical axis and/or an axis substantiallynormal the broad face 112. The focus actuator preferably enables precisecontrol of objective lens movement along the optical axis, such asenabling control to less than a threshold precision (e.g., 10, 50, 75,100, 150, 400, or 1000 nm). The second unit 142 can optionally includeoptical element selection actuators, such as rotational and/ortranslational actuators that move optical elements (e.g., objectivelenses, filters, etc.) into and/or out of the optical path. The secondunit 142 can additionally or alternatively include lateral translationactuators (e.g., enabling and/or controlling translation of imagingsubsystem elements along axes substantially parallel the broad face 112and/or perpendicular the optical axis), tilt actuators (e.g., enablingand/or controlling rotation of imaging subsystem elements, such as aboutaxes substantially parallel the broad face 112 and/or normal the opticalaxis), and/or any other suitable actuators.

The third unit 143 preferably includes one or more actuators (e.g.,insertion actuator) that enable and/or control out-of-plane motion(e.g., translation along one or more out-of-plane axes not substantiallyparallel the broad face 112) of the particle extractor 134 (e.g.,relative to the capture stage 110 and/or particle receptacle station150). The out-of-plane axis is preferably an axis substantially normalthe broad face 112 and/or the well apertures. However, the out-of-planeaxes can additionally or alternatively include a vertical axis, an axissubstantially parallel an axis defined by the particle extractor 134(e.g., defined by the void, such as a central axis of the capillarytube), an axis substantially parallel the optical axis, and/or any othersuitable axes. For example, the insertion actuator can control insertion(and/or removal) of the capture end 135 into the substrate 200 (e.g.,into the target well; on top of the target well, such as with thecapture end 135 in contact with the top surface of the well; etc.),thereby enabling extraction of the well contents (e.g., cell and/or cellcluster, such as a cell captured in single-cell format). The insertionactuator preferably enables precise control of particle extractor motionalong the out-of-plane axis (e.g., optical axis), such as enablingcontrol to less than a threshold precision (e.g., 10, 50, 75, 100, 150,400, or 1000 nm). The insertion actuator can additionally oralternatively be configured to use force sensing and/or stalling of theactuator motor (e.g., to allow precise positioning of the capture end135 on top of a nanowell).

The third unit 143 can additionally or alternatively include one or morelateral translation actuators. The lateral translation actuatorspreferably enable and/or control extractor 134 translation along one ormore axes substantially parallel the broad face 112 (e.g., the X- andY-axes) and/or substantially perpendicular the out-of-plane actuatoraxis. The lateral translation actuators can enable lateral alignment ofthe particle extractor 134, such as alignment with the optical axis, thetarget well, a particle receptacle 152 and/or portion thereof (e.g.,target well of a multi-well plate), and/or any other suitable element ofthe system. The third unit 143 can optionally include one or more tiltactuators, which can enable and/or control rotation of the extractor 134about one or more axes (e.g., lateral axes such as axes substantiallyparallel the X- and Y-axes). The tilt actuators can enable angularalignment of the particle extractor 134, can enable extraction ofparticles from wells with different orientations (e.g., includingorientations requiring insertion at oblique angles to the broad face112), and/or can perform any other suitable function.

The third unit 143 can additionally or alternatively include an actuator(e.g., analogous to the actuator described above regarding the firstunit 141) for moving the extractor 134 between the particle extractionconfiguration (e.g., in which the particle extractor 134 is aligned withthe particle capture substrate 200 and/or optical axis) and a particledelivery configuration (e.g., in which the particle extractor 134 isaligned with the particle receptacle station 150). For example, theextractor 134 (and optionally, other actuators of the third unit 143,the pump 132 and/or pump actuator 144, and/or any other suitableelements of the system) can translate along a track and/or rotate abouta joint axis (e.g., vertical axis, horizontal axis, etc.) of acantilever arm to switch between the particle extraction and particledelivery configurations. However, the third unit 143 can additionally oralternatively include any other suitable elements in any other suitableconfiguration.

The actuators of the third unit 143 preferably control motion of theparticle extractor 134 but not of the pump 132 (e.g., wherein theextractor 134 is mechanically coupled to the pump 132 by the actuators).However, all or some of the actuators of the third unit 143 canoptionally control motion of the pump 132 (e.g., moving the pump 132 andextractor 134 together).

The pump actuator 144 preferably functions to control pumping action(e.g., pressure differential, pumped volume, etc.) of the pump 132. Thepump actuator 144 can be used to control aspiration and/or delivery ofcell extractor contents (e.g., thereby enabling extraction of particles,such as cells, from the substrate 200 and/or delivery of the extractedparticles to the particle receptacle 152). In one example, the particleretriever subsystem 130 can include a linear actuator coupled to theplunger of the syringe pump and configured to translate the plungerwithin the barrel of the syringe pump (e.g, substantially along acentral axis defined by the barrel). The pump actuator 144 (e.g.,plunger linear actuator) is preferably controlled by a motor, but canadditionally or alternatively be manually actuated (e.g., by a knob)and/or controlled in any other suitable manner. In a second example, thepump actuator 144 includes a piezoelectric actuator (e.g., configured toperform pumping, such as by altering an internal volume of a positivedisplacement pump) configured to be controlled by electrical controlsignals (e.g., from the control subsystem 160). However, the actuationsubsystem 140 can include any other suitable pump actuators 144 of anyother suitable type, which can be controlled (e.g., manually,automatically, such as by the control subsystem 160, etc.) in anysuitable manner.

All or some of the actuators preferably enable precise (e.g.,sub-micron) control of system element movement. For example, theactuators can include micrometer heads and/or precision drives forprecise manual and/or motorized motion control. However, the actuatorscan additionally or alternatively include any other suitable actuatorswith any suitable precision. All or some of the actuators can optionallyinclude position detectors such as encoders (e.g., optical, magnetic,etc.; linear, rotary, etc.; absolute, relative, etc.), limit switches,and/or any other suitable position detectors. The position detectors arepreferably configured to sample position data and to communicate theposition data to other elements of the system (e.g., to the controlsubsystem 160, to servomotors, etc.). All or some of the actuators caninclude motors (e.g., stepper motors, servomotors, etc.) and/or anyother suitable mechanisms to enable automated control of the actuators(e.g., by the control subsystem 160).

In some variations, the actuation subsystem 140 (e.g., enabling controlof capture stage movement, imaging subsystem movement, particleretriever subsystem movement, and/or movement of any other suitableelements of the system) includes elements (and/or enables control) suchas described in U.S. application Ser. No. 15/430,833, filed 13 Feb. 2017and titled “System for Imaging Captured Cells”, which is hereinincorporated in its entirety by this reference (e.g., as describedregarding the platform, focusing and optics module, and/or any othersuitable elements). For example, the first unit 141 can includeactuators such as described regarding the platform, the second unit 142can include actuators such as described regarding the focusing andoptics module, and the third unit 143 and/or pump actuator 144 caninclude actuators analogous to those described regarding the actuatorsof the platform and/or focusing and optics module. However, theactuation subsystem 140 can additionally or alternatively include anyother suitable actuators.

In some variations, all or some actuators of the actuation subsystem 140can be configured to be controlled (e.g., be automatically controlled)by the control subsystem 160. For example, the control subsystem 160 canautomatically control the first unit 141 (e.g., in order to facilitateautomated functions including autofocusing of objects of interest,self-calibration, captured cell and/or particle receptacle alignmentwith the particle extractor 134, cell capture device interrogation, cellcapture device agitation, etc.), second unit 142 (e.g., in order tofacilitate automated functions including autofocusing of objects ofinterest, self-calibration, magnification selection, filter selection,field of view selection, etc.), third unit 143 (e.g., in order tofacilitate automated functions including captured cell and/or particlereceptacle alignment with the particle extractor 134, particle extractorinsertion and/or withdrawal, etc.), pump actuator 144 (e.g., in order tofacilitate automated functions including aspiration and/or delivery offluid within the particle extractor 134, particle extractor primingand/or cleaning, etc.), and/or any other suitable elements of theactuation subsystem 140. However, all or some actuators can additionallyor alternatively be semi-automatically controlled and/or manuallycontrolled, such that a user or other entity can manipulate the capturestage 110 in some manner (e.g., using knobs, dials, and/or micrometerheads mechanically coupled to the capture stage 110).

1.5 Particle Receptacle Station.

The particle receptacle station 150 preferably functions to receive andretain one or more particle receptacles 152, and can optionally alignthe particle receptacles 152 relative to the the particle retrieversubsystem 130 (e.g., the cell extractor 134), capture stage 110, imagingsubsystem 120 (e.g., the illumination module 110, focusing and opticssubsystem 128, optical sensor 126, etc.), and/or any other suitableelements of the system 100. For example, the particle receptacle station150 can support one or more particle receptacles 152 (e.g., retainedagainst the station 150 by gravity and/or by one or more fasteners suchas spring clips and/or screws pressing upon each receptacle 152;retained within the station 150 by an inward force exerted alongsidewalls of the receptacle 152, such as a compressive force from afriction fit within a rubberized receptacle and/or any other suitableelement of the station 150; etc.). The particle receptacles 152 caninclude tubes (e.g., conical tubes, standard PCR tubes, etc.),multi-well plates (e.g., 96 well plates), Petri dishes, and/or any othersuitable receptacles (e.g., receptacles configured to receive and/orcontain cells and/or other particles). However, the particle receptaclestation 150 can additionally or alternatively include any other suitableelements in any other suitable arrangement.

The particle receptacle station 150 can be rigidly coupled to thecapture stage 110 (e.g., as described above), rigidly coupled to thestructural frame 10, actuatably coupled (e.g., by one or more actuatorsof the actuation subsystem 140, such as by the actuators of the first,second, and/or third units, and/or by other actuators enablingindependent motion of the particle receptacle station 150) to thestructural frame 10 and/or any other suitable element of the system,and/or can be arranged within the system in any other suitable manner.

1.6 Control Subsystem.

The control subsystem 160 preferably functions to control systemoperation, such as enabling implementation (e.g., automated and/orsemi-automated execution) of the methods 300 described below.

The control subsystem 160 can include one or more: processors (e.g.,CPU, GPU, microprocessor, etc.), memory and/or data storage modules(e.g., Flash, RAM, hard disk drive, etc.), and/or any other suitablecomponents. The processing system is preferably mounted to thestructural frame 10, but can alternatively be mounted to any othersuitable component, and/or can be mechanically separate from the otherelements of the system 100 (e.g., can be connected to the system by adata connector, can communicate wirelessly with other components of thesystem, etc.).

The control subsystem 160 is preferably configured to communicate withand/or control other system elements, such as the imaging subsystem 120and/or actuation subsystem 140. For example, the control subsystem 160can be coupled (e.g., electrically coupled; otherwise coupled by acoupling capable of transmitting power, control signals, and/or data;etc.) to the optical sensor 126 (enabling activation of the opticalsensor 126 and/or receipt of data, such as image data, from the opticalsensor 126) and to one or more actuators of the actuation subsystem 140(e.g., enabling control of the actuators and/or receipt of data, such asposition data, from the actuation subsystem 140 position sensors).However, the control subsystem 160 can additionally or alternativelyinclude any other suitable components, be connected to any othersuitable elements of the system, and/or perform any other suitablefunctions.

1.7 Display.

The system 100 can optionally include a display 170. The display 170 ispreferably configured to communicate with the control subsystem 160(e.g., coupled to the control subsystem 160 by a data connection, suchas a video data cable; configured to wirelessly receive information fromthe control subsystem 160; etc.). The display 170 is preferablyconfigured to display one or more of: control parameters of the systemassociated with the control subsystem 160 and images derived from theimage dataset. For example, the display can show images (e.g., near-realtime image streams, such as live videos; previously captured images;etc.) captured by the imaging subsystem 120 and/or derivatives thereof,control parameters and/or other information related to system operation(e.g., presented as overlays on the images, presented separate fromand/or in place of images, etc.), and/or any other suitable information.The control parameters (e.g., information) presented on the screen(e.g., presented in overlays) can include: positions of system elements(e.g., coordinates, visual indications within and/or outside the imagefield of view, etc.); current and/or planned motion of system elements;cell identifications such as selected/non-selected cells, cell types(e.g., determined based on fluorescence microscopy data), etc.; targetwells for cell retrieval (e.g., from wells of the substrate 200) and/orreception (e.g., at wells of the receptacle 152); retrieval processsteps and/or status (e.g., “calibrating”, “priming”, “identifyingcells”, “retrieving cell 21 of 40”, “washing capillary tube”, etc.);and/or any other suitable information. However, the display 170 canadditionally or alternatively perform any other suitable function.

1.8 Containment Subsystem.

The containment subsystem 180 preferably functions to create a sterileenvironment for sample handling (e.g., isolating the system contents,such as the contents of the substrate 200 and/or receptacle 152, from anambient environment surrounding the containment subsystem 180). In afirst embodiment, the containment subsystem 180 is a sterile hood (e.g.,biological safety cabinet), wherein the other elements of the system 100(e.g., the structural frame 10 and attached subsystems) fit within thesterile hood. In this embodiment, the structural frame 10 preferably hasdimensions sufficiently small to enable facile placement in (andoptionally, removal from) a biological safety cabinet (e.g., less than10 inches tall×24 inches wide×30 inches deep), but can alternativelyhave any other suitable dimensions. In a second embodiment, thecontainment subsystem 180 envelopes the structural frame 10 and attachedcomponents (e.g., is attached directly to the exterior of the structuralframe 10. In one example (e.g., as shown in FIGURE XX), the containmentsubsystem 180 includes a hinged cover operable between a closedconfiguration, in which some or all elements of the system (e.g., thecapture stage 110 and particle retriever subsystem 130) are enclosed bythe cover, and an open configuration which enables user access to theotherwise-enclosed components (e.g., to enable placement and/or removalof system elements, such as particle capture substrates 200, particleextractors 134, etc.). However, the containment subsystem 180 canadditionally or alternatively include any other suitable components inany suitable arrangement.

1.9 Particle Capture Substrate.

The particle capture substrate 200 preferably defines a closed surface220 (e.g., bottom surface) and an open surface 230 (e.g., top surface).The surfaces are preferably broad faces opposing each other (e.g.,substantially parallel each other) across the substrate body. The opensurface 230 preferably defines a plane, such as a substrate top plane.The substrate 200 preferably defines a set of wells within the substratebody, each well of the set defining: an aperture (e.g., at the plane); abase arranged within the substrate body (e.g., between the aperture andthe closed surface 220); and a wall extending from the aperture to thebase. Further, the substrate 200 can define a plurality of channelswithin the substrate body, wherein some or all of the wells are fluidlycoupled to one or more adjacent wells one or more of the channels.During cell extractor aspiration at a target well (e.g., duringextraction of a cell captured in the target well), these channels canfacilitate fluid flow (e.g., convective currents) from adjacent wells,through the target well, and into the cell extractor. This fluid flowcan enable, facilitate, and/or urge the captured cell into the cellextractor. In a specific example, the particle capture substrate 200defines a hexagonal array (e.g., close-packed array) of hexagonal wellswith micron-scale width (e.g., 1-100 microns, such as 1, 5, 10, 15, 20,25, 30, 35, 40, 50, or 60 microns). In this specific example, the wellsare subdivided into hexagonal groups of seven wells, wherein the wellsof each hexagonal group are fluidly connected by channels, and theinter-group walls separating adjacent hexagonal groups do not allowfluid communication between the hexagonal groups (e.g., do not definechannels).

Embodiments, variations, and examples of the particle capture substrate200 are described in U.S. application Ser. No. 13/557,510 titled “CellCapture System and Method of Use” and filed on 25 Jul. 2012, U.S.application Ser. No. 14/289,155 titled “System and Method for Isolatingand Analyzing Cells” and filed on 28 May 2014, and U.S. application Ser.No. 15/422,222 titled “System and Method for Isolating and AnalyzingCells” and filed on 24 Feb. 2017, which are each incorporated in theirentireties by this reference. However, the particle capture substrate200 can additionally or alternatively include any other suitableelements in any suitable arrangement.

As shown in FIGS. 3A and 3B, the system 100 can optionally functionwithin (and/or complementary to) a platform for capturing particles froma sample in single-particle format, wherein the platform includes asample preparation portion operable to process a sample containing a setof particles of interest, and to transmit the processed sample through aparticle capture substrate 200 (e.g. microfluidic chip, cell capturesubstrate, etc.) for capturing the set of particles in single-particleformat (and/or in particle clusters). The particles (e.g., cells) canthen be retrieved in single-cell format in a viable state for furtherprocessing and/or analysis (e.g., in relation to diagnosticapplications). In a first embodiment, the system 100 and particlecapture platform are integrated (e.g., share a common stage forretaining the particle capture substrate 200), wherein the particlecapture substrate 200 remains in place in the particle capture platformduring both processed sample transmission and subsequent particleretrieval. In a second embodiment, following processed sampletransmission, the particle capture substrate 200 can be removed from theparticle capture platform and placed in the capture stage 110 forparticle retrieval. However, the system 100 and particle captureplatform can additionally or alternatively have any other suitablerelationship.

Embodiments, variations, and examples of the sample preparation portionare described in U.S. application Ser. No. 14/208,298 titled “System andMethod for Capturing and Analyzing Cells” and filed on 13 Mar. 2014,U.S. application Ser. No. 15/074,054 titled titled “System and Methodfor Capturing and Analyzing Cells” and filed on 18 Mar. 2016, and U.S.application Ser. No. 14/208,458 titled “System for Imaging CapturedCells” and filed on 13 Mar. 2014, which are each incorporated in theirentireties by this reference. However, the system 100 can additionallyor alternatively cooperate with any other suitable platform or platformcomponents.

2. Method.

A method 300 of captured particle retrieval preferably includes imagingcaptured particles (e.g., captured within a particle capture substrate200), selecting captured particles, extracting the selected particles,and delivering the extracted particles (e.g., as shown in FIG. 8). Theparticles are preferably cells (e.g., live cells), but can additionallyor alternatively include any other suitable particles. The method 300 ispreferably implemented using the system 100 (and/or particle captureplatform) described above, but can additionally or alternatively beimplemented using any other suitable mechanisms.

The captured particles are preferably imaged by the imaging subsystem120 (e.g., using bright-field microscopy, fluorescence microscopy,etc.). Particles are preferably selected (e.g., by the control subsystem160, by a user, etc.) based on the imaging (e.g., selecting a particulartype of cell, wherein the cell type is determined based on fluorescencemicroscopy). Particle extraction is preferably performed by the particleretriever subsystem 130, more preferably based on image data sampled bythe imaging subsystem 120 (e.g., live video showing capture end 135position relative to the selected cell), which can enable, for example,alignment of the particle extractor 134 over a target well andcontrolled insertion of the capture end 135 (e.g., into the target well;placement on top of the target well, such as with the capture end 135 incontact with the top surface of the walls defining the target well;etc.). Particle extraction is preferably performed by actuating the pump132 (e.g., to reduce pressure within the particle extractor, therebycausing aspiration) while the capture end 135 is inserted (e.g., intothe target well, on top of the target well, etc.). After extraction, theparticle extractor 134 is preferably repositioned at a target region(e.g., target well) of a particle receptacle 152, at which pointparticle delivery can be achieved by actuating the pump 132 (e.g., toincrease pressure within the particle extractor, thereby expelling itscontents).

The control subsystem 160 preferably enables automated (and/orsemi-automated) performance of the method 300 (and/or elements thereof).For example, the control subsystem 160 can be configured to perform(e.g., based on image data received from the imaging subsystem 120):automated focusing (e.g., by moving the objective lens) on imagingtargets such as wells, captured particles, and/or particle extractorcapture ends (e.g., capillary tip); automated identification of targetcells (e.g., based on fluorescence criteria); automated detection andlateral translation of the capture end (e.g., aligning the capture endwith a target well that contains a target cell, aligning the capture endwith a destination region of a particle receptacle, etc.); automatedplacement of the capture end in contact with (e.g., on top of, insertedinto, etc.) the target well (e.g., avoiding crashes which can damage thecapillary tip, rendering it inoperable to extract cells); automated pumpactuation (e.g., to effect aspiration and/or cell ejection); and/or anyother suitable elements of the method.

Placing the capture end in contact with the target well can include, forexample: focusing on a reference element of the capture substrate,preferably an element of the target well (e.g., top surface of thewell); focusing on the particle extractor (e.g., on the capture end,such as the capillary tip); determining a relative distance between thereference element and the particle extractor (e.g., based on theobjective lens motion required to switch focus between them); and movingthe particle extractor based on the relative distance (e.g., movingtoward the target well by an amount equal to the distance, moving by anamount less than the distance, etc.). In one example, focus can beadjusted (e.g., to follow the capture end movement, to switch back andforth between the reference element and the capture end, etc.) duringand/or between capture end movement (e.g., repeatedly), and the relativedistance determination can be updated accordingly. However, placing thecapture end in contact with the target well can additionally oralternatively be performed using any other suitable techniques (e.g.,insertion actuator force sensing and/or stalling).

The system 100 can additionally or alternatively support methods (e.g.,cell capture, imaging, and/or analysis methods) such as those describedin U.S. application Ser. No. 15/362,565, titled “System and Method forCapturing and Analyzing Cells” and filed 28 Nov. 2016, U.S. applicationSer. No. 14/208,298 titled “System and Method for Capturing andAnalyzing Cells” and filed on 13 Mar. 2014, U.S. application Ser. No.15/074,054 titled titled “System and Method for Capturing and AnalyzingCells” and filed on 18 Mar. 2016, and/or U.S. application Ser. No.14/208,458 titled “System for Imaging Captured Cells” and filed on 13Mar. 2014, which are each incorporated in their entireties by thisreference, and/or in any other suitable manner. For example, the method300 can include capturing particles (e.g., capturing live cells insingle-cell format) within wells of a particle capture substrate 200,prior to particle imaging, selection, extraction, and delivery (e.g., asshown in FIG. 9). However, the method 300 can additionally oralternatively include any other suitable elements performed in any othersuitable manner.

In one embodiment (e.g., as shown in FIG. 10), the method 300 includes:capturing live cells (e.g., in single-cell and/or single-cluster format)in a particle capture substrate 200; maintaining the cells in a viableformat (e.g., for multiple days, weeks, etc.); imaging the cells;processing the cells; re-imaging the cells after processing; selectingcells based on the imaging data (e.g., initial imaging and/orre-imaging); extracting the selected cells and delivering the extractedcells to a particle receptacle (e.g., using the system 100 as describedabove) such as a 96 well plate or a second particle capture substrate;maintaining and/or growing the cells (e.g., culturing the cells) for anextended time period (e.g., days, weeks, etc.); and/or imaging and/ormonitoring the extracted cells (e.g., during culturing). In a firstexample, processing the cells includes treating all captured cells witha set of reagents (e.g., CRISPR reagents). In a second example,processing the cells includes: selecting cells (e.g., a subset of thecells), such as based on the imaging data; and delivering reagents tothe selected cells (e.g., the same set of reagents for each selectedcell, different sets of reagents for different cells, etc.). In thisexample, the reagents can be delivered using the particle retrieversubsystem (e.g., using the cell extractor; using a different reagentdelivery element attached to the particle retriever subsystem, such asin place of the cell extractor; etc.) and/or any other suitable targeteddelivery mechanism. For example, the reagent can be delivered using athinner capillary tube (e.g, thin enough to fit inside the target well,thin enough to penetrate the captured cell, etc.) attached to theparticle retriever subsystem, and can optionally include inserting thecapillary tube into the target well and/or the captured cell for reagentdelivery (e.g., delivering CRISPR reagents directly into the cytosol ofthe target cell). However, the method 300 can additionally oralternatively include any other suitable elements.

The system 100 and method 300 of the preferred embodiment and variationsthereof can be embodied and/or implemented at least in part as a machineconfigured to receive a computer-readable medium storingcomputer-readable instructions. The instructions are preferably executedby computer-executable components preferably integrated with the systemand one or more portions of a processor and/or a controller. Thecomputer-readable medium can be stored on any suitable computer-readablemedia such as RAMs, ROMs, flash memory, EEPROMs, optical devices (CD orDVD), hard drives, floppy drives, or any suitable device. Thecomputer-executable component is preferably a general or applicationspecific processor, but any suitable dedicated hardware orhardware/firmware combination device can alternatively or additionallyexecute the instructions.

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of systems, methods and computer programproducts according to preferred embodiments, example configurations, andvariations thereof. In this regard, each block in the flowchart or blockdiagrams may represent a module, segment, or portion of code, whichcomprises one or more executable instructions for implementing thespecified logical function(s). It should also be noted that, in somealternative implementations, the functions noted in the block can occurout of the order noted in the FIGURES. For example, two blocks shown insuccession may, in fact, be executed substantially concurrently, or theblocks may sometimes be executed in the reverse order, depending uponthe functionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts, or combinations of special purpose hardware andcomputer instructions.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

We claim:
 1. A system for cell retrieval from a capture substratecomprising a set of wells distributed across a broad face of the cellcapture substrate, the system comprising: an imaging subsystemconfigured to sample an image data dataset of one or more of the set ofwells; a retriever subsystem comprising an extractor comprising anextractor tip, an outlet, and a void defined between the extractor tipand the outlet; an actuator configured to translate the extractor withrespect to the capture substrate, the actuator operable to place theextractor tip for fluid communication with a target well of the set ofwells; and an extraction subsystem configured to: receive the imagedataset; based on the image dataset, control the actuator to positionthe extractor tip for fluid communication with the target well; and inresponse to achieving positioning of the extractor tip for fluidcommunication with the target well, control the retriever subsystem toextract a target material from the target well.
 2. The system of claim1, wherein a cross section of each of the set of wells defines apolygon.
 3. The system of claim 1, wherein the capture substrate furthercomprises a manifold defining a manifold inlet and a manifold outlet,wherein the manifold inlet and the manifold outlet are fluidly coupledto each well in the set of wells.
 4. The system of claim 1, wherein theimaging system comprises: an optical sensor; a light source configuredto transmit light toward the target well; a lens defining an opticalaxis substantially normal the broad face, wherein the lens is configuredto perform at least one of focusing light onto the target material anddirecting light from the target material to the optical sensor; and afocus actuator configured to translate the lens substantially along theoptical axis.
 5. The system of claim 1, wherein the extractor comprisesa capillary tube configured for application of a capillary force to thetarget material.
 6. The system of claim 1, wherein the extractorcomprises a pump coupled to the outlet and configured to apply a removalforce in coordination with operation of the extraction subsystem.
 7. Thesystem of claim 6, wherein the extraction subsystem comprisesinstructions for repeated aspiration and delivery of content from and tothe target well.
 8. The system of claim 1, wherein the void of theextractor terminates at an aperture of the extractor tip, the aperturedefining a width between 10 microns and 50 microns.
 9. The system ofclaim 1, wherein the target material comprises at least one of a cell, afunctionalized particle, and content derived from a biological sample.10. The system of claim 1, further comprising a capture receptacle,wherein the extractor tip is operable to move the target material fromthe target well to the capture receptacle.
 11. A system for cellretrieval from a capture substrate comprising a set of wells distributedacross a broad face of the cell capture substrate, the systemcomprising: a retriever subsystem comprising an extractor comprising anextractor tip, an outlet, and a void defined between the extractor tipand the outlet; a positioning subsystem configured to position theextractor with respect to the capture substrate, the positioningsubsystem operable to place the extractor tip for communication with atarget well of the set of wells; and an extraction subsystem configuredto: guide the positioning subsystem to position the extractor tip forcommunication with at least one target well; and in response toachieving positioning of the extractor tip for fluid communication withthe target well, control the retriever subsystem to extract a targetmaterial from the at least one target well.
 12. The system of claim 11,wherein the target material comprises at least one of a cell, afunctionalized particle, and content derived from a biological sample.13. The system of claim 12, wherein a cross section of each of the setof wells defines a hexagon.
 14. The system of claim 13, wherein theextraction subsystem is configured for repeated aspiration and deliveryof content from and to the at least one target well.
 15. The system ofclaim 11, wherein the extractor comprises a capillary tube configuredfor application of a capillary force to the target material.
 16. Thesystem of claim 11, wherein the extractor comprises a pump coupled tothe outlet and configured to apply a removal force in coordination withoperation of the extraction subsystem.
 17. The system of claim 11,further comprising a substrate actuator comprising a platform, thesubstrate actuator configured to translate the capture substraterelative to the retriever subsystem.
 18. The system of claim 11, furthercomprising an imaging subsystem configured to sample an image datadataset of one or more of the set of wells, wherein the extractionsubsystem is further configured to receive the image dataset and basedon the image dataset, control the positioning subsystem to position theextractor tip for fluid communication with the target well.
 19. Thesystem of claim 18, wherein the imaging subsystem comprises: an opticalsensor; a light source configured to transmit light toward the targetwell; a lens defining an optical axis substantially normal the broadface, wherein the lens is configured to perform at least one of focusinglight onto the target material and directing light from the targetmaterial to the optical sensor; and a focus actuator configured totranslate the lens substantially along the optical axis.
 20. The systemof claim 11, wherein the set of wells is divided into a set of subsetsof wells, wherein each subset of wells is circumscribed by a closedcurve, and an inner diameter of the extractor tip is smaller than alargest chord of the closed cu